Reversible dimerization of a CLC transporter: A model for membrane protein foldin

摘要 蛋白质折叠的核心谜团在于自然界的物理力量如何驱动一串简单的氨基 酸转化成稳定的构象确定的蛋白质。对于可溶性蛋白质，疏水基团的埋藏 远离水界面是一个主要的驱动力，但膜包埋蛋白质不能经历 疏水力，因为脂质双层缺乏水。一个基本的难题由此产生： 油脂蛋白质表面在油脂脂质双层中找到它的油脂蛋白质伴侣，忠实地折叠成它的天然分子。 结构？最近，一种结构稳定和功能性单体形式的正常同型二聚体Cl-/H+ 通过在二聚体界面引入色氨酸突变来设计反向转运蛋白CLC-ec 1。初步 研究表明，蛋白质可以通过额外的突变或在某些脂质中转变回二聚体状态。 条件这些结果将CLC-ec 1作为研究可逆二聚化的模型， 简化了蛋白质折叠过程，同时仍然包含蛋白质的所有热力学性质 膜环境中的相互作用。为了进行这些能量测量，单体/二聚体 将使用三种成熟的方法对群体进行定量：（i）单体与单体的“泊松计数”。 脂质体群体中的二聚体，（ii）脂质体中的荧光自猝灭，以及（iii）Förster共振 能量转移（FRET）在脂质体和支持的双层单分子研究。通过这些检测 在适当的地方，将进行实验，以调查两个替代假设，已经弥漫 在这个领域的话语。第一，特异性跨膜螺旋相互作用是由货车德驱动的 高度互补表面上的瓦尔斯力。自由能的变化将在替换后测量 的界面残基丙氨酸或色氨酸，与显着的位置进一步研究，通过增加侧- 链体积来调节货车范德华相互作用。第二个假设是， 由螺旋缔合后脂质的熵增加驱动。为了研究这个，形成脂质的分子 溶剂将通过改变化学头基、链长和链序进行改性， 来自古细菌的不饱和或四醚脂质。对于所有实验，自由能关系也将是 相对于温度测量以外推焓和熵的值。这些结果将 提供了深入了解膜蛋白相互作用的驱动力，甚至可能提供一个基础， 用于解决蛋白质在脂质双层这种奇怪溶剂中折叠的基本问题。

Abstract The central enigma of protein folding lies in how the physical forces of nature drive a simple string of amino acids into a stable, conformationally defined protein. For soluble proteins, the burial of hydrophobic groups away from aqueous interfaces is a major driving force, but membrane-embedded proteins cannot experience hydrophobic forces, as the lipid bilayer lacks water. A fundamental conundrum thus arises: how does a greasy protein surface find its greasy protein partner in the greasy lipid bilayer to fold faithfully into its native structure? Recently, a structurally stable and functional monomeric form of the normally homodimeric Cl-/H+ antiporter CLC-ec1 was designed by introducing tryptophan mutations at the dimer interface. Preliminary studies show that the protein can be shifted back to the dimer state with additional mutations or in certain lipid conditions. These results present CLC-ec1 as a model for the study of reversible dimerization, which simplifies the protein folding process while still encompassing all of the thermodynamic properties of protein interactions in the membrane environment. To make these energetic measurements, the monomer/dimer populations will be quantified using three well-established methods: (i) "Poisson-counting" of monomer vs. dimers in liposome populations, (ii) fluorescence self-quenching in liposomes, and (iii) Förster resonance energy transfer (FRET) in liposomes and supported bilayers for single molecule studies. With these assays in place, experiments will be carried out to investigate two alternative hypotheses that have pervaded discourse in this field. First, that specific transmembrane helix interactions are enthalpy-driven by van der Waals forces at highly complementary surfaces. Changes in free energy will be measured upon substitution of interface residues to alanine or tryptophan, with significant positions studied further by increasing side- chain volume to modulate the van der Waals interactions. The second hypothesis is that interactions are driven by increased entropy of lipids upon helix association. To study this, the molecules forming the lipid solvent will be modified by changing the chemical head group, chain length and chain order using unsaturated or tetra-ether lipids from archaea. For all experiments, free energy relationships will also be measured with respect to temperature to extrapolate values for enthalpy and entropy. These results will provide insight into the driving forces for membrane protein interactions, and may even provide a foundation for attacking general questions underlying protein folding in the strange solvent that is the lipid bilayer.